catpb s  (Tocris)

Journal: Journal of leukocyte biology

Article Title: The ketone body acetoacetate activates human neutrophils through FFAR2.

doi: 10.1093/jleuko/qiad035

Figure Lengend Snippet: Fig. 1. Acetoacetate activates FFAR2 in HEK293 cells overexpressing the receptor. HEK293 cells overexpressing FFAR2 were activated with different concentrations of (A) propionate and (B) acetoacetate (AA), and cAMP was measured in the presence of 1 μM forskolin. Data obtained were fitted to dose–response curves (mean ± SD, n = 3 independent experiments), and EC50 values were calculated. EC50 was 410 μM (95% CI: 199 to 827 µM) for propionate and 1.24 mM (95% CI: 0.45 to 3.60 mM) for AA. (C) β-Arrestin translocation was measured continuously over time upon stimulation with acetoacetate 10 mM (closed triangles) and compared to background activity when no stimulus was added (open squares). The control shows the level of luminescence before addition of both stimuli and Nano-Glo reagent. (D) Bar graph showing the fold increase of peak values, compared to time-matched background activity, upon stimulation with propionate (black bar) and AA (white bars). The dotted line indicates the level of background activity. Data are presented as a mean ± SD (n = 3 individual experiments), and statistical analysis was performed using ratio paired t test (*P < 0.05, **P < 0.01).

Article Snippet: The FFAR2 antagonist CATPB ((S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)-phenyl) butanoic acid) was from Tocris (Bristol, UK), and the Gαq inhibitor YM-254890 was from WAKO (Wako Chemicals GmbH, Neuss, Germany).

Techniques: Translocation Assay, Activity Assay, Control

Journal: Journal of leukocyte biology

Article Title: The ketone body acetoacetate activates human neutrophils through FFAR2.

doi: 10.1093/jleuko/qiad035

Figure Lengend Snippet: Fig. 2. Acetoacetate triggers a transient rise in intracellular calcium ([Ca2+]i) in human neutrophils. (A, B) Human neutrophils were loaded with Fura-2 AM and stimulated with different concentrations of (A) propionate or (B) acetoacetate (AA). (C) Neutrophils were incubated with (dashed line) or without (solid line) the FFAR2 antagonist CATPB (100 nM) and then stimulated with AA (10 mM). (D, E) Neutrophils were incubated with (dashed line) and without (solid line) Cmp58 (1 μM) for 10 min and then stimulated with (D) propionate (25 µM) or (E) AA (3 mM). The rise in [Ca2+]i is presented as the ratio between Fura-2 fluorescence at 340 and 380 nm over time, and 1 representative experiment out of 3 independent experiments is shown.

Article Snippet: The FFAR2 antagonist CATPB ((S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)-phenyl) butanoic acid) was from Tocris (Bristol, UK), and the Gαq inhibitor YM-254890 was from WAKO (Wako Chemicals GmbH, Neuss, Germany).

Techniques: Incubation, Fluorescence

Journal: Journal of leukocyte biology

Article Title: The ketone body acetoacetate activates human neutrophils through FFAR2.

doi: 10.1093/jleuko/qiad035

Figure Lengend Snippet: Fig. 3. Acetoacetate triggers production of oxygen radicals in neutrophils allosterically modulated by Cmp58. (A, B) Neutrophils preincubated for 5 min in the absence (solid line) or presence (dashed line) of the FFAR2 allosteric modulator Cmp58 (1 μM) were stimulated with (A) propionate (25 µM) or (B) acetoacetate (AA, 10 mM), and superoxide release was measured over time. (C) Summary of the peak superoxide production (mean ± SD, n = 3 independent experiments) in response to stimulation with AA (10 mM) and propionate (25 μM) in cells treated with (white bars) or without (black bars) Cmp58 (1 μM). (D) Neutrophils treated with Cmp58 (1 μM) were stimulated with different concentrations of AA, and superoxide production was recorded. The peak values were determined, fitted to a dose–response curve (mean ± SD, n = 3 independent experiments), and the EC50 value was calculated. (E) Neutrophils were incubated for 5 min with Cmp58 (1 μM) together with (dashed line) or without (solid line) the FFAR2 antagonist CATPB (100 nM) prior to stimulation with AA (1 mM). (F) Bar graph (mean ± SD, n = 3 independent experiments) showing the peak superoxide production in cells incubated with Cmp58 (1 μM) together with (black bars) or without (white bars) CATPB (100 nM) and then stimulated with AA (1 mM) or propionate (25 μM). Statistical analysis in C and F was performed using a ratio paired t test (*P < 0.05, **P < 0.01).

Article Snippet: The FFAR2 antagonist CATPB ((S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)-phenyl) butanoic acid) was from Tocris (Bristol, UK), and the Gαq inhibitor YM-254890 was from WAKO (Wako Chemicals GmbH, Neuss, Germany).

Techniques: Incubation

Journal: Journal of leukocyte biology

Article Title: The ketone body acetoacetate activates human neutrophils through FFAR2.

doi: 10.1093/jleuko/qiad035

Figure Lengend Snippet: Fig. 4. Characterization of the interaction between acetoacetate and FFAR2. (A) Superoxide production by neutrophils preincubated for 5 min in the absence (dashed line) or presence (solid line) of acetoacetate (AA; 1 mM) before stimulation with Cmp58 (1 μM) and measurement of superoxide release over time. (B) Summary of the peak superoxide production (mean ± SD, n = 4 independent experiments) in response to stimulation with Cmp58 (1 μM) in cells pretreated with AA (1 mM) or propionate (25 μM) compared to the naive Cmp58 response. (C) Superoxide production by neutrophils preincubated for 5 min in the absence (dashed line) or presence (solid line) of AZ1729 (1 μM) before stimulation with AA (10 mM) and measurement of superoxide release over time. (D) The bar graph (mean ± SD, n = 3 independent experiments) shows the peak value of superoxide production by neutrophils pretreated for 5 min with (white bars) or without (black bars) AZ1729 (1 μM) prior to stimulation with AA (10 mM) or propionate (25 μM). (E) Neutrophils incubated with Cmp58 (1 μM) for 5 min were stimulated with either AA (10 mM, solid line) or buffer (dashed line) at the time point indicated by the left arrow. When the response had returned to basal levels, the neutrophils were restimulated with propionate (25 μM; time of addition shown by right arrow). (F) Summary of the peak superoxide production (mean ± SD, n = 3 independent experiments) induced by propionate or fMLF in cells first stimulated with AA (10 mM) followed by restimulation with either propionate (25 μM) or fMLF (100 nM). Cells treated with buffer (no additive) instead of AA were included as controls. Statistical analysis in B was performed using a repeated-measures 1-way ANOVA followed by Dunnett’s multiple comparison test, and for D and F, a ratio paired t test was performed (*P < 0.05; **P < 0.01).

Article Snippet: The FFAR2 antagonist CATPB ((S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)-phenyl) butanoic acid) was from Tocris (Bristol, UK), and the Gαq inhibitor YM-254890 was from WAKO (Wako Chemicals GmbH, Neuss, Germany).

Techniques: Incubation, Comparison

Journal: Journal of leukocyte biology

Article Title: The ketone body acetoacetate activates human neutrophils through FFAR2.

doi: 10.1093/jleuko/qiad035

Figure Lengend Snippet: Fig. 5. Acetoacetate activates allosterically modulated FFAR2 to produce extracellular ROS partially through Gαq signaling and trigger intracellular ROS production without NETosis. (A) Neutrophils preincubated for 5 min in the absence (white bars) or presence (black bars) of Gαq-specific inhibitor YM-254890 (200 nM) together with the FFAR2 allosteric modulator Cmp58 (1 μM) were stimulated with acetoacetate (AA, 10 mM), propionate (25 µM), or PAF (100 nM). The result is presented as remaining peak activity in response to stimulation comparing YM-254890 pretreated cells to untreated (mean ± SD, n = 3 individual experiments). (B) Intracellular production of oxygen radicals in response to AA (1 mM) was measured over time in neutrophils pretreated with (dotted line) and without (dashed line) Cmp58 (1 μM) for 5 min. Stimulation with phorbol myristate acetate (PMA; 50 nM) was performed in parallel as a reference (solid line). (C) Summary of the peak intracellular radical production (mean ± SD, n = 5 independent experiments) induced by AA (1 mM) in neutrophils preincubated with and without Cmp58 (1 μM) for 5 min. (D) Neutrophil NETosis (mean ± SD; n = 3 independent experiments) was determined by measuring levels of nucleic acid exposed extracellularly (using Sytox Green staining) over time in response to stimulation with PMA (50 nM, line with filled squares), AA (10 mM, line with triangles), and AA (10 mM) in presence of Cmp58 (1 μM, line with diamonds). The background level of fluorescence is included as a reference (no additive, line with open squares). Statistical analysis in A and C was performed by a ratio paired t test and for D by using a repeated-measures 1-way ANOVA followed by Dunnett’s multiple comparison test against no additive for values at time point 180 min (*P < 0.05, **P < 0.01).

Article Snippet: The FFAR2 antagonist CATPB ((S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)-phenyl) butanoic acid) was from Tocris (Bristol, UK), and the Gαq inhibitor YM-254890 was from WAKO (Wako Chemicals GmbH, Neuss, Germany).

Techniques: Activity Assay, Staining, Fluorescence, Comparison

Journal: Journal of leukocyte biology

Article Title: The ketone body acetoacetate activates human neutrophils through FFAR2.

doi: 10.1093/jleuko/qiad035

Figure Lengend Snippet: Fig. 6. Acetoacetate induces chemotaxis in neutrophils treated with FFAR2-positive allosteric modulators. (A) Neutrophil chemotaxis (n = 3 independent experiments) induced by different concentrations of propionate (white bars) or acetoacetate (AA; gray bars), fMLF included as a positive control (black bar, 10 nM). Data are presented as fold increase of spontaneous migration toward buffer (level of spontaneous migration is indicated with the dotted line). (B, C) Neutrophil chemotaxis (n = 3 independent experiments) induced by propionate (1 mM) and AA (5 and 1 mM) in the absence (white bars) or presence (gray bars) of either allosteric modulator (B) Cmp58 (1 μM) or (C) AZ1729 (1 μM). Data are presented as fold increase of migration toward the stimuli in the absence of allosteric modulator (level indicated by the dotted line). (D, E) Representative microscopic images of the neutrophils after allowing for migration for 90 min toward (D) AA (5 mM) or (E) AA (5 mM) in the presence of Cmp58 (1 μM). Statistical analysis in A was performed using a repeated-measures 1-way ANOVA followed by Dunnett’s multiple comparison test against buffer. A ratio paired t test was used for analysis of B and C (*P < 0.05, **P < 0.01; NS = no statistically significant difference).

Article Snippet: The FFAR2 antagonist CATPB ((S)-3-(2-(3-chlorophenyl)acetamido)-4-(4-(trifluoromethyl)-phenyl) butanoic acid) was from Tocris (Bristol, UK), and the Gαq inhibitor YM-254890 was from WAKO (Wako Chemicals GmbH, Neuss, Germany).

Techniques: Chemotaxis Assay, Positive Control, Migration, Comparison